Processing of the alaW alaX operon encoding the Ala2 tRNAs in Escherichia coli requires both RNase E and RNase P.

The alaW alaX operon encodes the Ala2 tRNAs, one of the two alanine tRNA isotypes in Escherichia coli. Our previous RNA-seq study showed that alaW alaX dicistronic RNA levels increased significantly in the absence of both RNase P and poly(A) polymerase I (PAP I), suggesting a role of polyadenylation in its stability. In this report, we show that RNase E initiates the processing of the primary alaW alaX precursor RNA by removing the Rho-independent transcription terminator, which appears to be the rate limiting step in the separation and maturation of the Ala2 pre-tRNAs by RNase P. Failure to separate the alaW and alaX pre-tRNAs by RNase P leads to poly(A)-mediated degradation of the dicistronic RNAs by polynucleotide phosphorylase (PNPase) and RNase R. Surprisingly, the thermosensitive RNase E encoded by the rne-1 allele is highly efficient in removing the terminator (>99%) at the nonpermissive temperature suggesting a significant caveat in experiments using this allele. Together, our data present a comprehensive picture of the Ala2 tRNA processing pathway and demonstrate that unprocessed RNase P substrates are degraded via a poly(A) mediated decay pathway.

Maturation of the E. coli Glu2, Ile1, and Ala1B tRNAs utilizes a complex processing pathway.

Despite significant progress in understanding the diversity of tRNA processing pathways in Escherichia coli, the mechanism for the maturation of tRNAs encoded within the rRNA operons has not received much attention. Here, we show that the Glu2, Ile1, and Ala1B tRNAs, encoded by 10 genes located between the 16S and 23S rRNAs in the seven rRNA operons, are matured via a RNase E-independent processing pathway that utilizes at least six different enzymes. It has been shown that the Glu2 and Ile1-Ala1B pre-tRNAs released by initial RNase III cleavages of the 30S primary rRNA transcripts retain extended 5'-leader (35-139 nt) and 3'-trailer (166-185 nt) sequences. However, the 5' maturation of the tRNAs by RNase P is inhibited until the trailer sequences are shortened to 1-4 nucleotides, initially by a second RNase III cleavage at 31-42 nucleotides downstream of the CCA determinant followed by exonucleolytic trimming. The RNase III cleaved Glu2 and Ile1-Ala1B trailer fragments are degraded via PAP I- dependent exonucleolytic decay. Compared to the six previously characterized tRNA processing pathways, maturation of the Glu2, Ile1, and Ala1B tRNAs is considerably more complex and appears to be distinct from what occurs in Gram-positive bacteria.

The C nucleotide at the mature 5' end of the Escherichia coli proline tRNAs is required for the RNase E cleavage specificity at the 3' terminus as well as functionality.

Proline tRNA 3'-maturation in Escherichia coli occurs through a one-step RNase E endonucleolytic cleavage immediately after the CCA determinant. This processing pathway is distinct from the 3'-end maturation of the other tRNAs by avoiding the widespread use of 3' → 5' exonucleolytic processing, 3'-polyadenylation and subsequent degradation. Here, we show that the cytosine (C) at the mature 5'-terminus of the proK and proL tRNAs is required for both the RNase E cleavage immediately after the CCA determinant and their functionality. Thus, changing the C nucleotide at the mature 5'-terminus of the proL and proK tRNAs to the more common G nucleotide led to RNase E cleavages 1-4 nucleotides downstream of the CCA determinant. Furthermore, the 5'-modified mutant tRNAs required RNase T and RNase PH for their 3'-maturation and became substrates for polyadenylation and degradation. Strikingly, the aminoacylation of the 5'-modified proline tRNAs was blocked due to the change in the recognition element for prolyl-tRNA-synthetase. An analogous modification of the pheV 5'-mature terminus from G to C nucleotide did not support cell viability. This result provides additional support for the importance of first nucleotide of the mature tRNAs in their processing and functionality.

Regulation of mRNA decay in E. coli.

Detailed studies of the Gram-negative model bacterium, Escherichia coli, have demonstrated that post-transcriptional events exert important and possibly greater control over gene regulation than transcription initiation or effective translation. Thus, over the past 30 years, considerable effort has been invested in understanding the pathways of mRNA turnover in E. coli. Although it is assumed that most of the ribonucleases and accessory proteins involved in mRNA decay have been identified, our understanding of the regulation of mRNA decay is still incomplete. Furthermore, the vast majority of the studies on mRNA decay have been conducted on exponentially growing cells. Thus, the mechanism of mRNA decay as currently outlined may not accurately reflect what happens when cells find themselves under a variety of stress conditions, such as, nutrient starvation, changes in pH and temperature, as well as a host of others. While the cellular machinery for degradation is relatively constant over a wide range of conditions, intracellular levels of specific ribonucleases can vary depending on the growth conditions. Substrate competition will also modulate ribonucleolytic activity. Post-transcriptional modifications of transcripts by polyadenylating enzymes may favor a specific ribonuclease activity. Interactions with small regulatory RNAs and RNA binding proteins add additional complexities to mRNA functionality and stability. Since many of the ribonucleases are found at the inner membrane, the physical location of a transcript may help determine its half-life. Here we discuss the properties and role of the enzymes involved in mRNA decay as well as the multiple factors that may affect mRNA decay under various in vivo conditions.

Inactivation of RNase P in Escherichia coli significantly changes post-transcriptional RNA metabolism.

Ribonuclease P (RNase P), which is required for the 5'-end maturation of tRNAs in every organism, has been shown to play a limited role in other aspects of RNA metabolism in Escherichia coli. Using RNA-sequencing (RNA-seq), we demonstrate that RNase P inactivation affects the abundances of ~46% of the expressed transcripts in E. coli and provide evidence that its essential function is its ability to generate pre-tRNAs from polycistronic tRNA transcripts. The RNA-seq results agreed with the published data and northern blot analyses of 75/83 transcripts (mRNAs, sRNAs, and tRNAs). Changes in transcript abundances in the RNase P mutant also correlated with changes in their half-lives. Inactivating the stringent response did not alter the rnpA49 phenotype. Most notably, increases in the transcript abundances were observed for all genes in the cysteine regulons, multiple toxin-antitoxin modules, and sigma S-controlled genes. Surprisingly, poly(A) polymerase (PAP I) modulated the abundances of ~10% of the transcripts affected by RNase P. A comparison of the transcriptomes of RNase P, RNase E, and RNase III mutants suggests that they affect distinct substrates. Together, our work strongly indicates that RNase P is a major player in all aspects of post-transcriptional RNA metabolism in E. coli.

Generation of pre-tRNAs from polycistronic operons is the essential function of RNase P in Escherichia coli.

Ribonuclease P (RNase P) is essential for the 5'-end maturation of tRNAs in all kingdoms of life. In Escherichia coli, temperature sensitive mutations in either its protein (rnpA49) and or RNA (rnpB709) subunits lead to inviability at nonpermissive temperatures. Using the rnpA49 temperature sensitive allele, which encodes a partially defective RNase P at the permissive temperature, we show here for the first time that the processing of RNase P-dependent polycistronic tRNA operons to release pre-tRNAs is the essential function of the enzyme, since the majority of 5'-immature tRNAs can be aminoacylated unless their 5'-extensions ≥8 nt. Surprisingly, the failure of 5'-end maturation elicits increased polyadenylation of some pre-tRNAs by poly(A) polymerase I (PAP I), which exacerbates inviability. The absence of PAP I led to improved aminoacylation of 5'-immature tRNAs. Our data suggest a more dynamic role for PAP I in maintaining functional tRNA levels in the cell.

New Insights into the Relationship between tRNA Processing and Polyadenylation in Escherichia coli.

Recent studies suggest that poly(A) polymerase I (PAP I)-mediated polyadenylation in Escherichia coli is highly prevalent among mRNAs as well as tRNA precursors. Primary tRNA transcripts are initially processed endonucleolytically to generate pre-tRNA species, which undergo 5'-end maturation by the ribozyme RNase P. Subsequently, a group of 3' → 5' exonucleases mature the 3' ends of the majority of tRNAs with few exceptions. PAP I competes with the 3' → 5' exonucleases for pre-tRNA substrates adding short poly(A) tails, which not only modulate the stability of the pre-tRNAs, but also regulate the availability of functional tRNAs. In this review, we discuss the recent discoveries of new tRNA processing pathways and the implications of polyadenylation in tRNA metabolism in E. coli.

Analysis of post-transcriptional RNA metabolism in prokaryotes.

Post-transcriptional RNA metabolic pathways play important roles in permitting prokaryotes to operate under a variety of environmental conditions. Although significant progress has been made during the last decade in deciphering RNA processing pathways in a number of bacteria, a complete understanding of post-transcriptional RNA metabolism in any single microorganism is far from reality. Here we describe multiple experimental approaches that can be used to study mRNA stability, tRNA and rRNA processing, sRNA metabolism, and polyadenylation in prokaryotes. The methods described here can be readily utilized in both Gram-negative and Gram-positive bacteria with simple modifications.

Genotoxic, Metabolic, and Oxidative Stresses Regulate the RNA Repair Operon of Salmonella enterica Serovar Typhimurium.

The σ54 regulon in Salmonella enterica serovar Typhimurium includes a predicted RNA repair operon encoding homologs of the metazoan Ro60 protein (Rsr), Y RNAs (YrlBA), RNA ligase (RtcB), and RNA 3'-phosphate cyclase (RtcA). Transcription from σ54-dependent promoters requires that a cognate bacterial enhancer binding protein (bEBP) be activated by a specific environmental or cellular signal; the cognate bEBP for the σ54-dependent promoter of the rsr-yrlBA-rtcBA operon is RtcR. To identify conditions that generate the signal for RtcR activation in S Typhimurium, transcription of the RNA repair operon was assayed under multiple stress conditions that result in nucleic acid damage. RtcR-dependent transcription was highly induced by the nucleic acid cross-linking agents mitomycin C (MMC) and cisplatin, and this activation was dependent on RecA. Deletion of rtcR or rtcB resulted in decreased cell viability relative to that of the wild type following treatment with MMC. Oxidative stress from peroxide exposure also induced RtcR-dependent transcription of the operon. Nitrogen limitation resulted in RtcR-independent increased expression of the operon; the effect of nitrogen limitation required NtrC. The adjacent toxin-antitoxin module, dinJ-yafQ, was cotranscribed with the RNA repair operon but was not required for RtcR activation, although YafQ endoribonuclease activated RtcR-dependent transcription. Stress conditions shown to induce expression the RNA repair operon of Escherichia coli (rtcBA) did not stimulate expression of the S Typhimurium RNA repair operon. Similarly, MMC did not induce expression of the E. colirtcBA operon, although when expressed in S Typhimurium, E. coli RtcR responds effectively to the unknown signal(s) generated there by MMC exposure.IMPORTANCE Homologs of the metazoan RNA repair enzymes RtcB and RtcA occur widely in eubacteria, suggesting a selective advantage. Although the enzymatic activities of the eubacterial RtcB and RtcA have been well characterized, the physiological roles remain largely unresolved. Here we report stress responses that activate expression of the σ54-dependent RNA repair operon (rsr-yrlBA-rtcBA) of S Typhimurium and demonstrate that expression of the operon impacts cell survival under MMC-induced stress. Characterization of the requirements for activation of this tightly regulated operon provides clues to the possible functions of operon components in vivo, enhancing our understanding of how this human pathogen copes with environmental stressors.

Enzymes Involved in Posttranscriptional RNA Metabolism in Gram-Negative Bacteria.

Gene expression in Gram-negative bacteria is regulated at many levels, including transcription initiation, RNA processing, RNA/RNA interactions, mRNA decay, and translational controls involving enzymes that alter translational efficiency. In this review, we discuss the various enzymes that control transcription, translation, and RNA stability through RNA processing and degradation. RNA processing is essential to generate functional RNAs, while degradation helps control the steady-state level of each individual transcript. For example, all the pre-tRNAs are transcribed with extra nucleotides at both their 5' and 3' termini, which are subsequently processed to produce mature tRNAs that can be aminoacylated. Similarly, rRNAs that are transcribed as part of a 30S polycistronic transcript are matured to individual 16S, 23S, and 5S rRNAs. Decay of mRNAs plays a key role in gene regulation through controlling the steady-state level of each transcript, which is essential for maintaining appropriate protein levels. In addition, degradation of both translated and nontranslated RNAs recycles nucleotides to facilitate new RNA synthesis. To carry out all these reactions, Gram-negative bacteria employ a large number of endonucleases, exonucleases, RNA helicases, and poly(A) polymerase, as well as proteins that regulate the catalytic activity of particular RNases. Under certain stress conditions, an additional group of specialized endonucleases facilitate the cell's ability to adapt and survive. Many of the enzymes, such as RNase E, RNase III, polynucleotide phosphorylase, RNase R, and poly(A) polymerase I, participate in multiple RNA processing and decay pathways.

The rph-1-Encoded Truncated RNase PH Protein Inhibits RNase P Maturation of Pre-tRNAs with Short Leader Sequences in the Absence of RppH.

RNase PH, encoded by the rph gene, is a 3'→5' exoribonuclease that in E. coli participates primarily in the 3' maturation of pre-tRNAs and the degradation of rRNA in stationary-phase cells. Interestingly, the routinely used laboratory strains of MG1655 and W3110 have naturally acquired the rph-1 allele, encoding a truncated catalytically inactive RNase PH protein which is widely assumed to be benign. Contrary to this assumption, we show that the rph-1-encoded Rph-1 protein inhibits RNase P-mediated 5'-end maturation of primary pre-tRNAs with leaders of <5 nucleotides in the absence of RppH, an RNA pyrophosphohydrolase. In contrast, RppH is not required for 5'-end maturation of endonucleolytically generated pre-tRNAs in the rph-1 strain and for any tRNAs in Δrph mutant or rph+ strains. We propose that the Rph-1 protein bound to the 3' end of the substrate creates a steric hindrance that in the presence of a triphosphate at the 5' end reduces the ability of RNase P to bind to the pre-tRNA.IMPORTANCE In this paper, we demonstrate that the rph-1 mutation found in commonly used E. coli strains leads to the synthesis of a truncated functionally inactive RNase PH protein that interferes with the 5'-end maturation of specific tRNAs with short 5' leaders by RNase P in the absence of RppH, an RNA pyrophosphohydrolase that converts primary 5' triphosphates into 5' monophosphates. The data presented indicate that the presence of the triphosphate interferes with RNase P binding to the pre-tRNA.

Regulation of mRNA Decay in Bacteria.

Gram-negative and gram-positive bacteria use a variety of enzymatic pathways to degrade mRNAs. Although several recent reviews have outlined these pathways, much less attention has been paid to the regulation of mRNA decay. The functional half-life of a particular mRNA, which affects how much protein is synthesized from it, is determined by a combination of multiple factors. These include, but are not necessarily limited to, (a) stability elements at either the 5' or the 3' terminus, (b) posttranscriptional modifications, (c) ribosome density on individual mRNAs, (d) small regulatory RNA (sRNA) interactions with mRNAs, (e) regulatory proteins that alter ribonuclease binding affinities, (f) the presence or absence of endonucleolytic cleavage sites, (g) control of intracellular ribonuclease levels, and (h) physical location within the cell. Changes in physiological conditions associated with environmental alterations can significantly alter the impact of these factors in the decay of a particular mRNA.

Endonucleolytic cleavages by RNase E generate the mature 3' termini of the three proline tRNAs in Escherichia coli.

We demonstrate here for the first time that proline tRNA 3' end maturation in Escherichia coli employs a one-step endonucleolytic pathway that does not involve any of the six 3' → 5' exonucleases (RNase T, RNase PH, RNase D, RNase BN, RNase II and polynucleotide phosphorylase [PNPase]) to generate the mature CCA terminus. Rather, RNase E is primarily responsible for the endonucleolytic removal of the entire Rho-independent transcription terminator associated with the proK, proL and proM primary transcripts by cleaving immediately downstream of the CCA determinant. In the absence of RNase E, RNase G and RNase Z are weakly able to process the proK and proM transcripts, while PNPase and RNase P are utilized in the processing of proL The terminator fragment derived from the endonucleolytic cleavage of proL transcript is degraded through a PNPase-dependent pathway. It is not clear which enzymes degrade the proK and proM terminator fragments. Our data also suggest that the mature 5' nucleotide of the proline tRNAs may be responsible for the cleavage specificity of RNase E at the 3' terminus.

RNase E-based degradosome modulates polyadenylation of mRNAs after Rho-independent transcription terminators in Escherichia coli.

Here we demonstrate that the RNase E-based degradosome is required for poly(A) polymerase I (PAP I)-dependent polyadenylation after Rho-independent transcription terminators for both mono- and polycistronic transcripts. Disruption of degradosome assembly in mutants lacking the polynucleotide phosphorylase (PNPase) binding domain led to a significant increase in the level of PNPase synthesized polynucleotide tails in the rpsJ and rpsM polycistronic transcripts and the lpp monocistronic transcript. The polynucleotide tails were mostly located within the coding sequences in the degradosome mutants compared to the wild type control where the majority of the PAP I synthesized poly(A) tails were after the Rho-independent transcription terminators. For the Rho terminated metNIQ operon, the tails for all three mRNAs were predominately polynucleotide and were located within the coding sequences in both wild type and degradosome mutant strains. Furthermore, by employing a pnp-R100D point mutant that encodes a catalytically inactive PNPase protein that still forms intact degradosomes, we show that a catalytically active PNPase is required for normal mRNA polyadenylation by PAP I. Our data suggest that polyadenylation requires a functional degradosome to maintain an equilibrium between free PNPase and the PAP I polyadenylation complex.

Processing of the seven valine tRNAs in Escherichia coli involves novel features of RNase P.

Here we report that RNase P is required for the initial separation of all seven valine tRNAs from three distinct polycistronic transcripts (valV valW, valU valX valY lysY and lysT valT lysW valZ lysY lysZ lysQ). Particularly significant is the mechanism by which RNase P processes the valU and lysT polycistronic transcripts. Specifically, the enzyme initiates processing by first removing the Rho-independent transcription terminators from the primary valU and lysT transcripts. Subsequently, it proceeds in the 3' → 5' direction generating one pre-tRNA at a time. Based on the absolute requirement for RNase P processing of all three primary transcripts, inactivation of the enzyme leads to a > 4-fold decrease in the levels of both type I and type II valine tRNAs. The ability of RNase P to initiate tRNA processing at the 3' ends of long primary transcripts by endonucleolytically removing the Rho-independent transcription terminator represents a previously unidentified function for the enzyme, which is responsible for generating the mature 5' termini of all 86 E. coli tRNAs. RNase E only plays a very minor role in the processing of all three valine polycistronic transcripts.

In vivo analysis of polyadenylation in prokaryotes.

Polyadenylation at the 3' ends of mRNAs, tRNAs, rRNAs, and sRNAs plays important roles in RNA metabolism in both prokaryotes and eukaryotes. However, the nature of poly(A) tails in prokaryotes is distinct compared to their eukaryotic counterparts. Specifically, depending on the organism, eukaryotic poly(A) tails average between 50 and >200 nt and can easily be isolated by several techniques involving oligo(dT)-dependent cDNA amplification. In contrast, the bulk of the poly(A) tails present on prokaryotic transcripts is relatively short (<10 nt) and is difficult to characterize using similar techniques. This chapter describes methods that can circumvent these problems. For example, we discuss how to isolate total RNA and characterize its overall polyadenylation status employing a poly(A) sizing assay. Furthermore, we describe a technique involving RNase H treatment of total RNA followed by northern analysis in order to distinguish length of poly(A) tails on various types of transcripts. Finally, we outline a useful procedure to clone the poly(A) tails of specific transcripts using 5'-3' end-ligated RNA, which is independent of oligo(dT)-dependent cDNA amplification. These approaches are particularly helpful in analyzing transcripts with either short or long poly(A) tails both in prokaryotes and eukaryotes.

Deregulation of poly(A) polymerase I in Escherichia coli inhibits protein synthesis and leads to cell death.

Polyadenylation plays important roles in RNA metabolism in both prokaryotes and eukaryotes. Surprisingly, deregulation of polyadenylation by poly(A) polymerase I (PAP I) in Escherichia coli leads to toxicity and cell death. We show here that mature tRNAs, which are normally not substrates for PAP I in wild-type cells, are rapidly polyadenylated as PAP I levels increase, leading to dramatic reductions in the fraction of aminoacylated tRNAs, cessation of protein synthesis and cell death. The toxicity associated with PAP I is exacerbated by the absence of either RNase T and/or RNase PH, the two major 3' → 5' exonucleases involved in the final step of tRNA 3'-end maturation, confirming their role in the regulation of tRNA polyadenylation. Furthermore, our data demonstrate that regulation of PAP I is critical not for preventing the decay of mRNAs, but rather for maintaining normal levels of functional tRNAs and protein synthesis in E. coli, a function for polyadenylation that has not been observed previously in any organism.

RNAsnap™: a rapid, quantitative and inexpensive, method for isolating total RNA from bacteria.

RNAsnap™ is a simple and novel method that recovers all intracellular RNA quantitatively (>99%), faster (<15 min) and less expensively (∼3 cents/sample) than any of the currently available RNA isolation methods. In fact, none of the bacterial RNA isolation methods, including the commercial kits, are effective in recovering all species of intracellular RNAs (76-5700 nt) with equal efficiency, which can lead to biased results in genome-wide studies involving microarray or RNAseq analysis. The RNAsnap™ procedure yields ∼60 µg of RNA from 10(8) Escherichia coli cells that can be used directly for northern analysis without any further purification. Based on a comparative analysis of specific transcripts ranging in size from 76 to 5700 nt, the RNAsnap™ method provided the most accurate measure of the relative amounts of the various intracellular RNAs. Furthermore, the RNAsnap™ RNA was successfully used in enzymatic reactions such as RNA ligation, reverse transcription, primer extension and reverse transcriptase-polymerase chain reaction, following sodium acetate/ethanol precipitation. The RNAsnap™ method can be used to isolate RNA from a wide range of Gram-negative and Gram-positive bacteria as well as yeast.

Polyadenylation helps regulate functional tRNA levels in Escherichia coli.

Here we demonstrate a new regulatory mechanism for tRNA processing in Escherichia coli whereby RNase T and RNase PH, the two primary 3' → 5' exonucleases involved in the final step of 3'-end maturation, compete with poly(A) polymerase I (PAP I) for tRNA precursors in wild-type cells. In the absence of both RNase T and RNase PH, there is a >30-fold increase of PAP I-dependent poly(A) tails that are ≤10 nt in length coupled with a 2.3- to 4.2-fold decrease in the level of aminoacylated tRNAs and a >2-fold decrease in growth rate. Only 7 out of 86 tRNAs are not regulated by this mechanism and are also not substrates for RNase T, RNase PH or PAP I. Surprisingly, neither PNPase nor RNase II has any effect on tRNA poly(A) tail length. Our data suggest that the polyadenylation of tRNAs by PAP I likely proceeds in a distributive fashion unlike what is observed with mRNAs.

Bacterial/archaeal/organellar polyadenylation.

Although the first poly(A) polymerase (PAP) was discovered in Escherichia coli in 1962, the study of polyadenylation in bacteria was largely ignored for the next 30 years. However, with the identification of the structural gene for E. coli PAP I in 1992, it became possible to analyze polyadenylation using both biochemical and genetic approaches. Subsequently, it has been shown that polyadenylation plays a multifunctional role in prokaryotic RNA metabolism. Although the bulk of our current understanding of prokaryotic polyadenylation comes from studies on E. coli, recent limited experiments with Cyanobacteria, organelles, and Archaea have widened our view on the diversity, complexity, and universality of the polyadenylation process. For example, the identification of polynucleotide phosphorylase (PNPase), a reversible phosphorolytic enzyme that is highly conserved in bacteria, as an additional PAP in E. coli caught everyone by surprise. In fact, PNPase has now been shown to be the source of post-transcriptional RNA modifications in a wide range of cells of prokaryotic origin including those that lack a eubacterial PAP homolog. Accordingly, the past few years have witnessed increased interest in the mechanism and role of post-transcriptional modifications in all species of prokaryotic origin. However, the fact that many of the poly(A) tails are very short and unstable as well as the presence of polynucleotide tails has posed significant technical challenges to the scientific community trying to unravel the mystery of polyadenylation in prokaryotes. This review discusses the current state of knowledge regarding polyadenylation and its functions in bacteria, organelles, and Archaea.